SUMMARY The parent R37 project employs biophysical (electrophysiology, optical imaging, photometry), biochemical, pharmacological, genetic and cell biological approaches to understand an important new paradigm regarding the regulation of cellular bioenergetics that involves constitutive Ca2+ delivery from the endoplasmic reticulum to mitochondria mediated by Ca2+ release through the InsP3R Ca2+-release channel and Ca2+ uptake by the mitochondrial Ca2+-uniporter complex. A key experimental approach in our Aims that is employed daily is quantitative low-light level fluorescence microscopy of single living cells and intracellular organelles. Recently, the 18-year old computer that controls image acquisition, peripheral devices and image analysis, crashed irretrievably. Although we were fortunate to have an 18-year old backup computer that has enabled us to reboot the hardware, key features of the software are no longer functional in this computer. The system is so old that it is no longer supported by the original vendor. Accordingly, much of our research is in imminent jeopardy of a computer crash that will leave us unable to perform our proposed studies. We are requesting funds to replace the crippled 18-year old microscope and imaging system. We plan to image indicator dyes and genetically-encoded indicators in cytoplasm, mitochondria and endoplasmic reticulum in single cells over extended periods. This requires high resolution and low toxicity. Spinning-disk confocal microscopy is ideal for our needs. Accordingly, this request is for funds to purchase a BioVision Technologies, Inc. custom spinning disk confocal microscope. We anticipate that the instrument will be used daily by multiple investigators in the lab over a several year period, coincident with the extended funding of the R37.